Helicobacter pylori activates calpain via toll-like receptor 2 to disrupt adherens junctions in human gastric epithelial cells

Helicobacter pylori is a risk factor for the development of gastritis, gastroduodenal ulcers, and gastric adenocarcinoma. H. pylori-induced disruption of epithelial adherens junctions (AJs) is thought to promote the development of severe disease; however, the mechanisms whereby H. pylori alters AJ structure remain incompletely understood. The present study demonstrates that H. pylori infection in human patients is associated with elevated serum levels of an 80-kDa E-cadherin ectodomain, whose presence is independent of the presence of serum antibodies against CagA. In vitro, a heat-labile H. pylori surface component activates the host protease calpain in human gastric MKN45 cells independently of the virulence factors CagA and VacA. H. pylori-induced calpain activation results in cleavage of E-cadherin to produce a 100-kDa truncated form and induce relocalization of E-cadherin and β-catenin. Stimulation of MKN45 cells with the toll-like receptor 2 (TLR2) ligand P3C activated calpain and disrupted E-cadherin and β-catenin in a pattern similar to that induced by H. pylori. Inhibition of TLR2 prevented H. pylori-induced calpain activation and AJ disassembly. Together, these findings identify a novel pathway whereby H. pylori activates calpain via TLR2 to disrupt gastric epithelial AJ structure.     

Effects of Helicobacter pylori on the cadherin-catenin complex

BACKGROUND: The cadherin-catenin complex is the key component of the adherens junction in epithelial cells, and changes in this complex are implicated in gastric adenocarcinoma. Germline mutations in E-cadherin have been described in diffuse-type gastric adenocarcinoma. Helicobacter pylori infection is the first stage in gastric carcinogenesis. AIMS: To determine whether H pylori was associated with changes in the complex, and whether this was affected by virulence of the strain. METHODS: Epithelial cell lines were cultured with H pylori using the wild-type pathogenic and non-pathogenic strains and CagE null and VacA null isogenic mutants. Gastric biopsy specimens at endoscopy were obtained from patients with (n = 17) and without (n = 15) H pylori infection, and E-cadherin and beta-catenin expression was assessed by immunohistochemistry. H pylori was typed by polymerase chain reaction from these patients for CagE and VacA. RESULTS: In vitro studies showed that coculture with a pathogenic strain of H pylori led to disruption of epithelial junctional beta-catenin expression, but without evidence of nuclear translocation or signalling. This effect was independent of a functional Cag pathogenicity island and vacuolating activity, but dependent on live bacteria. No marked differences in beta-catenin or E-cadherin expression were seen in gastric biopsy specimens in patients with and without H pylori infection. CONCLUSION: Acute H pylori infection disrupts junctional beta-catenin in vitro, but chronic infection by H pylori has no effect on E-cadherin and beta-catenin expression, as seen in gastric biopsy specimens at the initial gastritis stage of the proposed Correa pathway of gastric carcinogenesis. A later effect at the later stages of atrophy or intestinal metaplasia cannot be ruled out.     

体外诱导脂肪来源间充质干细胞间质-上皮转换

目的探讨在体外诱导人脂肪来源间充质干细胞(hAD-MSCs)发生间质-上皮转换(MET)的可行性。方法从人脂肪中分离、培养间充质干细胞,流式细胞术鉴定其细胞表面标志。在体外添加激活素A(activin A)、维甲酸(RA)和骨形态发生蛋白7(BMP7)培养后,倒置显微镜下观察hAD-MSCs诱导后的形态变化,采用RT-PCR方法检测间质和上皮细胞相关基因vimentin,E-cadherin,ZO-1和β-catenin在诱导分化过程中表达水平改变。Western blot检测诱导前后间质标记vimentin和上皮标记β-catenin的表达。结果诱导后细胞形态发生改变,由长梭形变为卵圆形,上皮标记基因E-cadherin,ZO-1和β-catenin的mRNA表达显著上调(P<0.05),同时间质标志基因vimentin的mRNA表达显著下降(P<0.05)。Western blot结果显示,与未诱导组相比,诱导后细胞开始表达β-catenin,而vimentin的表达明显下降(P<0.05)。结论 Activin A、RA和BMP7联合使用可诱导hAD-MSCs发生MET的过程。     

Dickkopf-1沉默通过下调β-catenin水平抑制胃癌细胞侵袭及上皮-间充质转化

目的:探讨分泌蛋白Dickkopf-1(DKK1)在人胃癌细胞中的表达及其对胃癌细胞侵袭能力的影响。方法:以real-time PCR和Western blot法检测DKK1在人胃黏膜细胞(GES-1)和胃癌细胞(MKN-45和SGC-7901)中的表达水平;以RNA干扰法沉默DKK1,沉默效果以real-time PCR、Western blot及ELISA法验证;Transwell实验检测细胞侵袭能力,以丝裂霉素C抑制细胞增殖;real-time PCR及Western blot法检测细胞E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(vimentin)及β-连环蛋白(β-catenin)水平。结果:DKK1在MKN-45和SGC-7901细胞中的表达明显高于GES-1细胞,表明DKK1在胃癌细胞中表达显著升高;DKK1在MKN-45和SGC-7901细胞中被成功沉默后,细胞侵袭能力显著下降,并具有时间依赖性,同时伴随E-cadherin表达增高及N-cadherin和vimentin表达下降,表明DKK1沉默能够显著抑制胃癌细胞侵袭和上皮-间充质转化(epithelial-mesenchymal transition,EMT);经外源性重组DKK1(r DKK1)转染肿瘤细胞后,进一步证实DKK1具有促胃癌细胞侵袭的作用,并可促进EMT进程;DKK1沉默通过下调β-catenin水平来实现其对胃癌细胞侵袭及EMT的抑制作用。结论:DKK1在人胃癌细胞中表达显著增高,且DKK1沉默能够通过下调β-catenin水平抑制胃癌细胞侵袭及EMT过程。     

CIP2A过表达对胃黏膜上皮细胞增殖和凋亡的影响

目的:探究蛋白磷酸酶2A癌性抑制因子(CIP2A)过表达对胃黏膜上皮细胞GES-1增殖和凋亡的影响。方法:RT-q PCR检测正常胃黏膜组织和胃息肉组织中CIP2A和cyclin D1的表达。将胃黏膜上皮GES-1细胞分为空白对照组、空载体对照组和CIP2A过表达组,分组感染GES-1细胞后,分别用MTT法和Brd U试剂盒检测细胞增殖能力,流式细胞术检测细胞的凋亡情况,Western blot法和RT-q PCR检测凋亡蛋白的表达,ELISA检测细胞培养上清炎性因子的浓度。用E2F1 siRNA转染细胞,检测各组细胞中p-Rb、E2F1和cyclin D1的蛋白水平。结果:与正常胃黏膜组织相比,腺瘤性胃息肉组织中CIP2A和cyclin D1的mRNA表达水平均明显升高,而增生性胃息肉中CIP2A和cyclin D1的水平并无明显变化。GES-1细胞感染CIP2A过表达重组腺病毒之后,细胞的增殖能力和细胞活力提高,细胞凋亡率降低,炎性因子IL-1β和IL-10分泌增加,p-Rb、E2F1和cyclin D1蛋白水平升高,而E2F1沉默降低p-Rb、E2F1和cyclin D1的蛋白水平。结论:CIP2A通过激活Rb/E2F1促进GES-1细胞的增殖并抑制凋亡。     

IL-22诱导胃上皮细胞释放炎症因子并促进淋巴细胞趋化

为探讨IL-22在胃部炎症中的作用,我们采用流式细胞术和组织免疫荧光染色检测胃上皮细胞系AGS细胞及胃组织中IL-22受体的表达;采用IL22刺激胃上皮细胞系AGS及GES-1细胞,定量PCR检测其S100钙结合蛋白(S100)A8、S100A9、IL-8、基质金属蛋白酶(MMP)-1、MMP-10的表达;通过Transwell细胞趋化试验检测IL-22对淋巴细胞的趋化作用。结果发现,胃上皮细胞系及胃组织中有IL-22R1表达,并且IL-22能够诱导胃上皮细胞产生炎症因子及MMP,某些因子可促进淋巴细胞趋化。据此说明IL-22通过调控胃上皮细胞产生炎症因子并趋化淋巴细胞浸润,参与胃部炎症反应。     

Down-regulation of a morphogen (sonic hedgehog) gradient in the gastric epithelium of Helicobacter pylori-infected Mongolian gerbils

Sonic hedgehog (Shh) is a morphogen involved in many aspects of patterning of the gut during embryogenesis and in gastric fundic gland homeostasis in the adult. Intestinal metaplastic change of the gastric epithelium is associated with the loss of Shh expression, and mice that lack Shh expression show intestinal transformation of the gastric mucosa. The present study was designed to investigate the alteration of Shh expression in the stomach of an experimental model of Helicobacter pylori (H. pylori) colonization. Male Mongolian gerbils were inoculated with H. pylori and examined 4 and 51 weeks later. The level of Shh mRNA expression was determined by quantitative RT-PCR and in situ hybridization. Shh protein expression was determined by immunoblotting and immunohistochemistry. Shh was expressed in the parietal cells, zymogenic cells, and mucous neck cells of the gastric fundic glands of gerbils. Prolonged colonization by H. pylori led to extension of the inflammation from the antrum to the corpus of the stomach, with loss of Shh expression. Loss of Shh expression correlated with loss of parietal cells, disturbed maturation of the mucous neck cell-zymogenic cell lineage, and increased cellular proliferation. Shh expression is significantly reduced in H. pylori-associated gastritis. These data show for the first time that H. pylori infection leads to down-regulation of the expression of a morphogen with an established role (Shh) in gastric epithelial differentiation.     

Effects of Nonsteroidal Anti-Inflammatory Drugs on Helicobacter pylori-Infected Gastric Mucosae of Mice: Apoptosis, Cell Proliferation, and Inflammatory Activity

Helicobacter pylori and nonsteroidal anti-inflammatory drugs (NSAIDs) are two well-known important causative factors of gastric damage. While H. pylori increases apoptosis and the proliferation of gastric epithelial cells and is an important factor in peptic ulcer and gastric cancer, NSAIDs induce cell apoptosis and have antineoplastic effects. We investigated the effects of NSAIDs (a nonselective cyclooxygenase [COX] inhibitor [indomethacin] and a selective COX-2 inhibitor [NS-398]) on the apoptosis and proliferation of gastric epithelial cells and gastric inflammation in H. pylori-infected mice. C57BL/6 mice were sacrificed 8 weeks after H. pylori SS1 inoculation. Indomethacin (2 mg/kg) or NS-398 (10 mg/kg) was administered subcutaneously once daily for 10 days before sacrifice. The following were assessed: gastric inflammatory activity, gastric COX protein expression by Western blotting; gastric prostaglandin E(2) levels by enzyme immunoassay, apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, and cell proliferation by Ki67 immunostaining. Compared to the controls, H. pylori infection and/or NSAID treatment increased COX-1 and COX-2 protein expression. Gastric prostaglandin E(2) levels, apoptotic index, cell proliferation index, neutrophil activity, and the degree of chronic inflammation were all increased by H. pylori infection, and these effects were significantly decreased by indomethacin treatment. However, NS-398 treatment after H. pylori infection did not induce a significant reduction, although it did result in a tendency to decrease. These results show that NSAIDs can reverse the increased apoptosis and proliferation of epithelial cells and inflammatory activity in the stomachs of H. pylori-infected mice and that, like COX-2 activation, COX-1 induction contributes to the change of gastric mucosal cell turnover and inflammation induced by H. pylori infection.     

Effect of bone morphogenetic protein-2 on proliferation and apoptosis of gastric cancer cells

Objective: To investigate the effects of bone morphogenetic protein-2 (BMP-2) on the proliferation, differentiation and apoptosis of normal human gastric mucosal cells and gastric cancer cells.Methods: Poorly differentiated gastric cancer BGC823 cells, moderately differentiated gastric cancer cells and normal human gastric mucosal epithelial GES-1 cells were independently treated with recombinant human BMP-2 or its inhibitor Noggin. MTT assay was performed to detect the proliferation, flow cytometry done to measure the cell cycle and apoptosis and immunohistochemistry carried out to determine the expression of cyclin-dependent kinase 4 (CDK4).Results: BMP-2 exerted inhibitory effect on the growth of all types of cells and the inhibition become more evident with the increase of BMP-2 dose. After treatment with 200 ng/ml BMP-2, cancer cells arrested in G1 phase and those in S phase reduced. Gastric cancer cells had higher CDK4 expression than GES-1 cells. BMP-2 decreased CDK-4 expression in cancer cells but had no influence in GES-1 cells. Noggin conferred promotive effect on the growth of 3 types of cells. In 2 types of cancer cells, treatment with 2000 ng/ml Noggin significantly increased the proportion of cells in S phase but reduced that in G1 phase. However, Noggin did not affect the cell cycle of GES-1 cells. The CDK4 expression was markedly increased in 2 types of cancer cells but that of GES-1 remained unchanged after treatment with 2000 ng/ml Noggin.Conclusions: BMP-2 may inhibit the proliferation of both normal and malignant gastric epithelial cells, down-regulate CDK4 expression in gastric cancer cells and arrest gastric cancer cells in G1-phase in cell cycle. Through antagonizing BMP-2, Noggin, may accelerate the proliferation of gastric cancer cells. Thus, the abnormality of BMP signaling pathway may play an important role in the pathogenesis of gastric cancer.     

Heat shock protein 70 is involved in polaprezinc driven cell protection against Helicobacter pylori-induced injury

Polaprezinc (PZ) plays a role in the protection of gastric mucosa and inhibiting Helicobacter pylori (H. pylori) growth in vitro. The objective of this study was to determine the protective effects of PZ on human gastric epithelial cells (GES-1) against H. pylori-induced damage, while also examining heat shock protein 70 (HSP70) as a potential underlying factor in this protection. Our findings revealed that PZ exerted bactericidal effects against H. pylori strains. We also observed that PZ mitigated the H. pylori-induced damage to GES-1 cells by increasing cell viability, reducing LDH release, and decreasing the secretion of pro-inflammatory factors such as MCP-1 and IL-6. Co-culturing PZ with GES-1 cells significantly up-regulated the GES-1 HSP70 expression in both a time and dose-dependent manner. Pre-incubating (for 12 h) or co-culturing (for 24 h) GES-1 cells with PZ reversed the down-regulation of HSP70 in GES-1 cells caused by H. pylori infection. However, when quercetin was used to inhibit the up-regulation of HSP70 in GES-1 cells, the protective effect of PZ on GES-1 cells was significantly reduced. Based on the results of this study, PZ exhibits a protective role on GES-1 cells against H. pylori injury, as well as a direct bactericidal effect on H. pylori. HSP70 is involved in the PZ-driven host cell protection against H. pylori injury. These findings provide insight into alternative strategies for H. pylori treatment.     

纺锤菌素抑制胃癌细胞的侵袭和转移

目的探讨纺锤菌素(netropsin)对胃癌细胞侵袭和转移能力的影响及其分子机制。方法用Transwell检测胃癌细胞侵袭和转移能力,用Western blot检测EMT相关标志物E-cadherin和vimentin表达,用免疫荧光检测β-catenin的细胞定位,验证netropsin作用前后Wnt/β-catenin信号通路活性。结果 Netropsin浓度25μmol/L时对MKN28细胞增殖有抑制作用,netropsin可以降低上皮标志物E-cadherin的表达,上调间质标志物vimentin的表达;netropsin可以通过抑制胃癌细胞的EMT,降低胃癌细胞侵袭和转移能力(P0.05),同时可阻止β-catenin进入细胞核。结论 Netropsin可以通过与HMGA2竞争结合转录因子结合位点抑制Wnt/β-catenin信号通路,降低胃癌细胞EMT的发生,从而抑制胃癌细胞侵袭能力。     

Augmented gp130-mediated cytokine signalling accompanies human gastric cancer progression

H. pylori infection accounts for most cases of gastric cancer, but the initiating events remain unclear. The principal H. pylori pathogenicity-associated CagA protein disrupts intracellular SHP-2 signalling pathways including those used by the IL-6 family cytokines, IL-6 and IL-11. Imbalanced IL-6 family cytokine signalling in the gp130(757FF) mouse model of gastric cancer arising from hyperactivation of oncogenic STAT3 after altered SHP-2 : ERK1/2 signalling produces dysplastic antral tumours preceded by gastritis and metaplasia. In a cohort of patient gastric biopsies with known H. pylori and CagA status, we investigated whether (i) STAT3 and ERK1/2 activation is altered in H. pylori-dependent gastritis; (ii) these profiles are more pronounced in CagA+ H. pylori infection; and (iii) the expression of pro-inflammatory cytokines that activate STAT3 and ERK 1/2 pathways is associated with progression to gastric cancer. IL-6, IL-11, and activated STAT3 and ERK1/2 were quantified in antral biopsies from gastritic stomach, metaplastic tissue, and resected gastric cancer tissues. We observed significantly increased STAT3 and ERK1/2 activation (p = 0.001) in H. pylori-dependent gastritis, which was further enhanced in the presence of CagA+ H. pylori strains. Of known gastric ligands that drive STAT3 activation, IL-6 expression was increased after H. pylori infection and both IL-6 and IL-11 were strongly up-regulated in the gastric cancer biopsies. This suggests a mechanism by which IL-11 drives STAT3 activation and proliferation during gastric cancer progression. We addressed this using an in vitro approach, demonstrating that recombinant human IL-11 activates STAT3 and concomitantly increases proliferation of MKN28 gastric epithelial cells. In summary, we show increased STAT3 and ERK1/2 activation in H. pylori-dependent gastritis that is likely driven in an IL-6-dependent fashion. IL-11 expression is associated with adenocarcinoma development, but not gastritic lesions, and we identify a novel mechanism for IL-11 as a potent inducer of proliferation in the human gastric cancer setting.     

Expression of the programmed death ligand 1, B7-H1, on gastric epithelial cells after Helicobacter pylori exposure promotes development of CD4+ CD25+ FoxP3+ regulatory T cells

During Helicobacter pylori infection, T cells are recruited to the gastric mucosa, but the host T-cell response is not sufficient to clear the infection. Some of the recruited T cells respond in a polarized manner to a Th1 response, while others become anergic. We have previously shown that T-cell anergy may be induced during infection by the interaction of T cells with B7-H1, which is up-regulated on the gastric epithelium during H. pylori infection. Recently, regulatory T (Treg) cells with a CD4(+) CD25(high) FoxP3(+) phenotype were found at an increased frequency in the gastric mucosa of biopsy specimens from H. pylori-infected patients. While Treg cells are important in maintaining tolerance, they can also suppress immune responses during infection. In this study, we examined the induction of the Treg phenotype when na?ve T cells were incubated with gastric epithelial cells exposed to H. pylori. The frequency of this phenotype was markedly decreased when B7-H1 was blocked with monoclonal antibodies or its expression was blocked with small interfering RNA. The functional role of these Treg cells was assessed in proliferation assays when the cells were cocultured with activated T cells, which effectively decreased proliferation of the cells.     

幽门螺杆菌诱导人胃上皮细胞(GES-1)自噬的研究

 摘要:目的 观察幽门螺杆菌感染人胃上皮细胞后自噬的发生及变化情况,为进一步研究幽门螺杆菌感染后诱发自噬反应的相关研究奠定基础。方法 采用中性红摄入法确定H. pylori感染细胞的最佳浓度,以此浓度分别感染细胞,通过Western Blot检测感染不同时间LC3与p62的表达情况,免疫荧光染色检测自噬体。使用自噬抑制剂3MA、E64d、 pepstatin A干预后,观察感染后自噬变化情况。结果 H. pylori (22695) 感染GES-1细胞最佳浓度为m.o.i=400。感染后2小时即可观察到自噬的发生,且呈时间依赖性,于8小时到达高峰,24小时开始恢复基础水平。使用3MA阻断自噬,自噬体明显减少,且细胞形态欠佳。使用E64d和pepstatin A处理后自噬也受到一定程度的抑制。结论 H. pylori（22695）能够诱导人胃上皮（GES-1）发生自噬,且可被自噬抑制剂所抑制。     

LMP1 antagonizes WNT/β-catenin signalling through inhibition of WTX and promotes nasopharyngeal dysplasia but not tumourigenesis in LMP1(B95-8) transgenic mice

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) can induce cell transformation and tumourigenesis, but the mechanism is not understood. Previous studies have suggested that LMP1 acts through up-regulation of cellular proliferation pathways including the Wnt/β-catenin pathway, in which β-catenin is the central effector. Increased levels of β-catenin coupled with a decrease in E-cadherin lead to reduced cell adhesion. This pathway is antagonized by WTX (Wilms' tumour gene on the X chromosome), which can promote the ubiquitination and degradation of β-catenin. In the present study, we established L2/LMP1B(95 - 8) /EGFP transgenic mice to investigate the in vivo role of LMP1. Down-regulation of WTX and E-cadherin was accompanied by increased expression of β-catenin in these mice. Even though invasive tumours did not develop, dysplasia was seen in the nasopharynx and oropharynx epithelium of these transgenic mice. Analysis of LMP1(+) , WTX(+) , and LMP1 siRNA silenced HNE-1 cell lines demonstrated that WTX could exert a dominant role in LMP1-mediated WNT/β-catenin pathway regulation. This study indicates that LMP1 antagonizes the WNT/β-catenin pathway by inhibiting WTX, and this reduction in WTX is associated with epithelial dysplasia via regulation of the WNT/β-catenin pathway molecules E-cadherin and β-catenin. Further studies are required for a better understanding of the relationship between LMP1-mediated antagonization of the WNT/β-catenin pathway and tumourigenesis.